Isolation of Rabbit Cardiomyocytes
Stephen C Armstrong, Ph.D., Asst Professor,
Dept of Pathology,
East Tenn. State Univ,
Johnson City TN ,
Olaf Oldenburg, M.D.
Dept of Physiology
University of South Alabama
We have experience isolating rat, rabbit and pig cardiomyocytes. Each of these animal models have their distinct advantages and disadvantages and these must be taken into account when choosing the species for your model. The factors to consider include: Labor intensivity of the isolation procedure, cost efficiency, cell yield and relevance to the human model. From my experience, rabbit cardiomyocytes are a good compromise and balance of these logistical and scientific considerations. Rats are cheaper and pigs give a high cell yield and are potentially more relevant to the human model. However, the isolation of rabbit cardiomyocytes is relatively inexpensive and is not as labor intensive as the isolation of pig cardiomyocytes, delivering a higher cell yield than the rat model. The rabbit model does not share the distinction of the rat model, in which adenosine does not mimic cardioprotection. The rabbit is intermediate between the rat and pig in terms of heart rate and action potential duration and by these criteria the pig is more closely related to the human. We have found distinctions between the rabbit and pig cardiomyocytes in terms of their responses to our in vitro models of ischemia and the delay of osmotic fragility by ischemic preconditioning(IPC). The onset of osmotic fragility is defined as the sarcolemmal rupture and inability to exclude trypan blue after a brief resuspension of ischemic cells in a hypotonic (85mOsm) buffer. Osmotic fragility has been proposed to be a correlate of irreversible ischemic injury1-3. After 60 min. of ischemia, rabbit cardiomyocytes contract concurrent with ATP depletion and osmotic fragility occurs 30-60 min. after the onset of cell contracture. The onset of osmotic fragility in pig cardiomyocytes occurs concurrent with cell contracture 4. In contrast to rabbits, in which in vitro IPC does not alter cell contracture, subjection of pig cardiomyocytes to a IPC protocol, protects cells by delaying cell contracture and thus the onset of osmotic fragility. I will present the details of our cell isolation methods and our in vitro models of ischemia and ischemic preconditioning.
Isolated, calcium tolerant, adult rabbit cardiomyocytes are prepared by collagenase perfusion as previously described 5according to the methods of Hohl et. al. 6, with the specific modification that adenosine is excluded from all media. Our calcium-free perfusion buffer contains in mM, NaCl (125), MgSO4 (1.18), KCl (4.75), KH2PO4 (1.2 ), HEPES (30) bovine serum albumin (BSA, fraction V) (1g/liter), glucose (11), taurine (58.5) creatine (24.9) EGTA (0.02)*, plus a complete amino acid mixture (MEM Amino Acid Solution (Gibco, 20ml/L of 50x stock) and MEM Non-Essential Amino Acid Solution (Gibco,10ml/L of 100x stock) and vitamin solution (Sigma, 10ml/L of 100x stock). The mixture is bubbled with a 95% O2 / 5% CO2 mixture for 30 min and then the pH is adjusted to 7.2 before filtering. This perfusion buffer is then filtered through a 5 µm cellulose acetate filter. You will need about a liter for each isolation.
Our anesthesia consists of 50 mg/ml pentobarbital I.V. in animal that have been heparinzed with 1000U/Kg heparin I.P., 10 min prior to anesthesia. The thoracic cavity is entered and hearts are excised and placed in 200 ml of ice cold perfusion buffer and the aorta is quickly isolated and mounted on a Langendorff apparatus. The heart is flushed for 5 min in a non-recirculating mode with 37º C oxygenated (95% O2/5% CO2) perfusion buffer. The heart is perfused at 80 - 100 cm H20 by gravity.
After a 5 min equilibration period perfusion is switched to a recirculating collagenase perfusion using a peristaltic pump. The perfusate consists of 200 ml of the above described perfusion buffer to which 200 U/ml collagenase (Worthington, Type II) is added and then filtered through a 5 µm cellulose acetate filter. We use Worthington Type II collagenase and several lots are tested periodically to determine which lot will give the ideal preparation of cells (samples free of charge are available from Worthington Inc.). Even if these enzymes are called "collagenase" they consists of a mixture of collagenase and caseinase, and have clostripain and tryptic activities. In general I prefer collagenase lots that have relatively low activities of collagenase (200-250 U/mg). After switching to the collagenase perfusion, pH (7.3 7.4) and coronary pressure are continuously monitored. Adjust as needed with NaOH. After the collagenase reacts with the heart for 3-5 min there is a fall in the coronary pressure and the flow rate should be increased to restore the pressure to 80 -100 cmH2O. Perfusion is continued for 15-20 minutes until the heart starts to fall apart.
Ventricles are then removed and minced in 50ml of collagenase perfusate containing to which BSA has been added to achieve a final concentration of 2%. Disperse the cells by flushing them in and out of a large bore pipette (e. g. a plastic transfer pipette from which the tip has been cut off) 10 to 15 times. Cells are incubated in this mincing buffer for 10 min using a shaker bath at 37° C and bubbled with 95% O2 / 5% CO2. The next step is a nylon mesh filtration (200 350m M pore diameter). Cells are pelleted by a 1.5 min 200 rpm centrifugation (for this use four 15ml tubes with 12.5ml cell suspension in each) and then resuspended in 20 ml of wash buffer, containing in mM, NaCl (125), KCl (4.75), KH2PO4 (1.2), MgSO4 (1.2), HEPES (30), glucose (11), and 2% BSA supplemented with creatine, taurine, vitamins and amino acids as above but without EGTA or Ca2+. You need about 100ml of this buffer for the purification steps. Filter the wash buffer through a 5 µm cellulose acetate filter. Cells are incubated in this wash buffer for 30 minutes in a 100 ml Erlenmeyer flask in a 37°C shaker water bath at 25 shakes/min to allow re-establishment of normal electrolyte gradients. Supply oxygen by gently blowing 95% O2 / 5% CO2 over the surface. The 30 min incubation is followed by addition of five aliquots of 100ul of 50 mM CaCl stock solution at 5 min intervals to achieve a final concentration of 1.25 mM.
For purification of viable calcium tolerant cells, divide the cell suspension equally into two 15ml tubes. Leave those tubes on the bench top for approximately 5min to get an initial sedimentation pellet, discard the supernatant. This step is followed by two brief centrifugations of approximately 200 rpm, 1 1.5 min each. After each spin discard the supernatant and resuspend the pellet into fresh calcium-containing wash buffer (add calcium to the above wash buffer to achieve 1.25mM Ca2+; pH 7.4), gently mix the cells with the fresh buffer before each centrifugation. Isolates averaged 45 million cells, 75-80 percent of which are rod-shaped (see figure 1).
Oxygenated cardiomyocytes are elongated with prominent striations indicating the relaxed state of sarcomeres . These cells exclude trypan blue and do not have blebs.
Ischemia model and assessment of cell injury:
An aliquot of the cell suspension is placed in a 1.8 ml microcentrifuge tube (Fisher Scientific, Pittsburgh, PA) and centrifuged into a pellet. Each cell pellet occupies a volume of about 0.25 ml, and measures 0.8-1 cm in thickness. Excess supernatant is removed to leave a fluid layer above the pelleted cells of about one third the volume of the pellet. After layering with mineral oil, the cell pellets were incubated without agitation at 37°C. A 25 µl sample of the final cell pellet is removed through the oil layer, with care to prevent introduction of air, at the appropriate time points and resuspended for 3-5 minutes in 200 µl of hypotonic (85 mOsm) buffer containing 3 mM amytal as a mitochondrial inhibitor, to preclude cell rounding due to reoxygenation during cell swelling. A 25 µl sample is mixed with an equal volume of counting media (0.5% glutaraldehyde in 85 mOsM modified Tyrodes solution, with reduced NaCl, containing 1% trypan blue). Microscopic examination at 100X magnification determined the morphology (rod, round or square (See Figure 2) and the permeability of the cells to trypan blue (See Figure 3) as described previously7, 8.
Ischemic contracture of rabbit cardiomyocytes occurs after 60 minutes of ischemia, concurrent with ATP depletion. Dome shaped, sub-sarcolemmal blebs are observed at this point of ischemia following resuspension in hypotonic buffer.
At 120 minutes of ischemia, osmotic fragility of cardiomyocytes is observed by the inability of cells to exclude trypan blue after resuspension in a hypotonic buffer.
High levels of ATP in oxygenated rabbit cardiomyocytes can be indirectly assessed by the mitochondrial potential fluorescent indicator, Rhodamine 123.
ATP depletion is observed in the contracted (square shaped) , ischemic cells.
Immuno-staining of oxygenated cardiomyocytes for vinculin shows a costameric (rib-like) pattern at the sarcolemma and staining at the intercalated disks.
Immuno-staining of ischemic cardiomyocytes for vinculin shows that the level of fluorescence at the sarcolemma and intercalated disk is diminished 9.
Transmission electron microscopy of oxygenated rabbit cardiomyocytes demonstrates the relaxed state of the sarcomeres and dense, unswollen mitochondria, similar to oxygenated myocardium. The nuclear chromatin is dispersed and the sarcolemma does not show blebs.
1. Steenbergen CJ, Hill ML, Jennings RB. Volume regulation and plasma membrane injury in aerobic, anaerobic and ischemic myocardium in vitro : Effect of osmotic swelling on plasma membrane integrity. Circ Res 1985;57:864-875.
2. Tranum-Jensen J, Janse MJ, Fiolet JWT, Krieger JG, D'Alnoncourt CN, Durrer D. Tissue osmolality, cell swelling, and reperfusion in acute regional myocardial ischemia in the isolated porcine heart. Circ Res 1981;49:364-381.
3. Ganote CE, Vander Heide RS. Irreversible injury of isolated adult rat myocytes: Osmotic fragility during metabolic inhibition. Am J Path 1988;132:212-222.
4. Armstrong SC, Kao R, Gao W, et al. Comparison of in vitro preconditioning responses of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin. J Mol Cell Cardiol 1997;29:3009-3024.
5. Vander Heide RS, Angelo JP, Altschuld RA, Ganote CE. Energy dependence of contraction band formation in perfused hearts and isolated adult myocytes. Am J Path 1986;125:55-68.
6. Hohl CM, Altschuld RA, Brierley GP. Effects of calcium on the permeability of isolated adult rat cells to sodium and potassium. Arch Biochem Biophys 1982;221:197-205.
7. Armstrong SC, Ganote CE. Effects of 2,3-butanedione monoxime (BDM) on contracture and injury of isolated rat myocytes following metabolic inhibition and ischemia. J Mol Cell Cardiol 1991;23:1001-1014.
8. Vander Heide RS , Rim D, Hohl CM, Ganote CE. An in vitro model of myocardial ischemia utilizing isolated adult rat myocytes. J. Mol Cell Cardiol 1990; 22:165-181
9. Armstrong, SC and Ganote CE. Flow cytometric analysis of isolated adult cardiomyocytes: Vinculin and tubulin fluorescence during metabolic inhibition and ischemia. J Mol Cell Cardiol 1992; 24: 149-162
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